Here we demonstrate that type I protein kinase A is redoxactive, forming an interprotein disulfide bond between its two regulatory RI subunits in response to cellular hydrogen peroxide. This oxidative disulfide formation causes a subcellular translocation and activation of the kinase, resulting in phosphorylation of established substrate proteins. The translocation is mediated at least in part by the oxidized form of the kinase having an enhanced affinity for ␣-myosin heavy chain, which serves as a protein kinase A (PKA) anchor protein and localizes the PKA to its myofilament substrates troponin I and myosin binding protein C. The functional consequence of these events in cardiac myocytes is that hydrogen peroxide increases contractility independently of ␤-adrenergic stimulation and elevations of cAMP. The oxidant-induced phosphorylation of substrate proteins and increased contractility is blocked by the kinase inhibitor H89, indicating that these events involve PKA activation. In essence, type I PKA contains protein thiols that operate as redox sensors, and their oxidation by hydrogen peroxide directly activates the kinase.There is now substantial evidence that oxidant species such as H 2 O 2 are produced in a regulated way in cells where they can function as signaling agents (1, 2). We have been studying the post-translational modification of protein cysteinyl thiols, as this is a major mechanism by which oxidants can alter the structure of proteins and so regulate their function. Our strategy has been to search for proteins that are susceptible to a variety of different modes of cysteine oxidation, such as S-thiolation (3, 4), sulfenation (5), and protein-protein disulfide bond formation (6). The rationale is that once we identify proteins with reactive thiols, the possibility that their oxidation has a functional correlate of physiological significance can be investigated. We previously found the RI regulatory subunits of protein kinase A (PKA) 2 form interprotein disulfide dimers during cardiac oxidative stress (6).Here we investigated the potential impact of this disulfide dimer formation on the function of PKA. PKA has two major forms (type I and type II), both of which exist as a tetramer comprising two catalytic and two regulatory subunits. There are two types of regulatory subunits (RI and RII), the presence of which in the PKA holokinase nominally defines the enzyme as type I or II, respectively. Recent studies have shown that the full dissociation of type I PKA in response to cAMP requires the presence of a substrate (7). This substrateinduced sensitization of type I PKA is not a feature of the type II enzyme (8). The regulatory subunits contain N-terminal sequences that are important for protein kinase A anchor protein (AKAP) binding. AKAPs are a diverse group of proteins that are found next to PKA substrate proteins and, thus, function to target PKA (9). Type I PKA is located in the cytosol, whereas type II is not as a result of being primarily bound (targeted) to AKAP proteins that are associated ...

The aim of the present study was to assess age-dependent changes of proteins in the vastus lateralis muscle of physically active elderly and young subjects by a combination of two-dimensional difference gel electrophoresis, SDS-PAGE and ESI-MS/MS. The differences observed in the elderly group included down-regulation of regulatory myosin light chains, particularly the phosphorylated isoforms, a higher proportion of myosin heavy chain isoforms 1 and 2A, and enhanced oxidative and reduced glycolytic capacity.

We show that proteomic analysis can be applied to study cartilage pathophysiology. Proteins secreted by articular cartilage were analyzed by two-dimensional SDS-PAGE and mass spectrometry. Cartilage explants were cultured in medium containing [ 35 S]methionine/cysteine to radiolabel newly synthesized proteins. To resolve the cartilage proteins by two-dimensional electrophoresis, it was necessary to remove the proteoglycan aggrecan by precipitation with cetylpyridinium chloride. 50 -100 radiolabeled protein spots were detected on two-dimensional gels of human cartilage cultures. Of 170 silverstained proteins identified, 19 were radiolabeled, representing newly synthesized gene products. Most of these were known cartilage constituents. Several nonradiolabeled cartilage proteins were also detected. The secreted protein pattern of explants from 12 osteoarthritic joints (knee, hip, and shoulder) and 14 nonosteoarthritic adult joints were compared. The synthesis of type II collagen was strongly up-regulated in osteoarthritic cartilage. Normal adult cartilage synthesized little or no type II collagen in contrast to infant and juvenile cartilage. Potential regulatory molecules novel to cartilage were identified; pro-inhibin ␤A and processed inhibin ␤A (which dimerizes to activin A) were produced by all the osteoarthritic samples and half of the normals. Connective tissue growth factor and cytokine-like protein C17 (previously only identified as an mRNA) were also found. Activin induced the tissue inhibitor for metalloproteinases-1 in human chondrocytes. Its expression was induced in isolated chondrocytes by growth factors or interleukin-1. We conclude that type II collagen synthesis in articular cartilage is down-regulated at skeletal maturity and reactivated in osteoarthritis in attempted repair and that activin A may be an anabolic factor in cartilage. Osteoarthritis (OA)1 is a common joint disease characterized by degeneration of articular cartilage. Since cartilage has very limited capacity for repair, the loss is effectively irreversible. Prevalence studies show that most people over the age of 65 have some evidence of the disease (1, 2). Little is known about the molecular mechanism of cartilage destruction in OA, particularly the early events. It is thought that there is an imbalance between anabolism and catabolism of the extracellular matrix, there being an increase in catabolism. It has been suggested that this increased breakdown of matrix is due to the production of degradative enzymes such as the matrix metalloproteinases (MMPs) and members of the disintegrin and metalloproteinase (ADAM) family (3, 4). The increase in proteinase expression may be due to inflammatory cytokines such as interleukin-1 (Il-1) and tumor necrosis factor (4, 5). However, it is unclear whether these degradative processes are a primary event or a secondary reaction.Articular cartilage consists mainly of extracellular matrix, the principal organic components of which are type II collagen fibers and aggregates of the large proteoglycan...

Regulation of protein function by reversible cysteinetargeted oxidation can be achieved by multiple mechanisms, such as S-glutathiolation, S-nitrosylation, sulfenic acid, sulfinic acid, and sulfenyl amide formation, as well as intramolecular disulfide bonding of vicinal thiols. Another cysteine oxidation state with regulatory potential involves the formation of intermolecular protein disulfides. We utilized two-dimensional sequential non-reducing/reducing SDS-PAGE (diagonal electrophoresis) to investigate intermolecular protein disulfide formation in adult cardiac myocytes subjected to a series of interventions (hydrogen peroxide, S-nitroso-Nacetylpenicillamine, doxorubicin, simulated ischemia, or metabolic inhibition) that alter the redox status of the cell. More detailed experiments were undertaken with the thiol-specific oxidant diamide (5 mM), a concentration that induces a mild non-injurious oxidative stress. This increase in cellular oxidation potential caused global intermolecular protein disulfide formation in cytosolic, membrane, and myofilament/cytoskeletal compartments. A large number of proteins that undergo these associations were identified using liquid chromatography-mass spectrometry/mass spectrometry. These associations, which involve metabolic and antioxidant enzymes, structural proteins, signaling molecules, and molecular chaperones, were confirmed by assessing "shifts" on non-reducing immunoblots. The observation of widespread protein-protein disulfides indicates that these oxidative associations are likely to be fundamental in how cells respond to redox changes.

Glutathione disulfide (GSSG) accumulates in cells under an increased oxidant load, which occurs during neurohormonal or metabolic stimulation as well as in many disease states. Elevated GSSG promotes protein S-glutathiolation, a reversible post-translational modification, which can directly alter or regulate protein function. We developed novel strategies for the study of protein S-glutathiolation that involved the simple synthesis of N,N-biotinyl glutathione disulfide (biotin-GSSG). Biotin-GSSG treatment of cells mimics a defined component of oxidative stress, namely a shift in the glutathione redox couple to the oxidized disulfide state. This induces widespread protein S-glutathiolation, which was detected on non-reducing Western blots probed with streptavidin-horseradish peroxidase and imaged using confocal fluorescence microscopy and ExtrAvidin-FITC. S-Glutathiolated proteins were purified using streptavidin-agarose and identified using proteomic methods. We conclude that biotin-GSSG is a useful tool in the investigation of protein S-glutathiolation and offers significant advantages over conventional methods or antibody-based strategies. These novel approaches may find widespread utility in the study of disease or redox signaling models where GSSG accumulation occurs. Molecular & Cellular Proteomics 5:215-225, 2006.

Here, we present the first study of a human neuromuscular disorder at transcriptional and proteomic level. Autosomal dominant facio-scapulo-humeral muscular dystrophy (FSHD) is caused by a deletion of an integral number of 3.3-kb KpnI repeats inside the telomeric region D4Z4 at the 4q35 locus. We combined a muscle-specific cDNA microarray platform with a proteomic investigation to analyse muscle biopsies of patients carrying a variable number of KpnI repeats. Unsupervised cluster analysis divides patients into three classes, according to their KpnI repeat number. Expression data reveal a transition from fast-glycolytic to slow-oxidative phenotype in FSHD muscle, which is accompanied by a deficit of proteins involved in response to oxidative stress. Besides, FSHD individuals show a disruption in the MyoD-dependent gene network suggesting a coregulation at transcriptional level during myogenesis. We also discuss the hypothesis that D4Z4 contraction may affect in trans the expression of a set of genes involved in myogenesis, as well as in the regeneration pathway of satellite cells in adult tissue. Muscular wasting could result from the inability of satellite cells to successfully differentiate into mature fibres and from the accumulation of structural damages caused by a reactive oxygen species (ROS) imbalance induced by an increased oxidative metabolism in fibres.

Clostridium difficile is a bacterium that causes disease of the large intestine, particularly after treatment with antibiotics. The bacterium produces two toxins (A and B) that are responsible for the pathology of the disease. In addition, a number of bacterial virulence factors associated with adhesion to the gut have previously been identified, including the cell wall protein Cwp66, the high-molecular weight surface layer protein (HMW-SLP) and the flagella. As the genome sequence predicts many other cell wall associated proteins, we have investigated the diversity of proteins in cell wall extracts, with the aim of identifying further virulence factors. We have used a number of methods to remove the proteins associated with the cell wall of C. difficile. Two of the resulting extracts, obtained using low pH glycine treatment and lysozyme digestion of the cell wall, have been analysed in detail by two-dimensional electrophoresis and mass spectrometry. One hundred and nineteen spots, comprising 49 different proteins, have been identified. The two surface layer proteins (SLPs) are the most abundant proteins, and we have also found components of the flagellum. Interestingly, we have also determined that a number of paralogs of the HMW-SLP are expressed, and these could represent targets for further investigation as virulence factors.

Protein sulfenic acids are reactive intermediates in the catalytic cycles of many enzymes as well as the in formation of other redox states. Sulfenic acid formation is a reversible post-translational modification with potential for protein regulation. Dimedone (5,5-dimethyl-1,3-cyclohexanedione) is commonly used in vitro to study sulfenation of purified proteins, selectively "tagging" them, allowing monitoring by mass spectrometry. However dimedone is of little use in complex protein mixtures because selective monitoring of labeling is not possible. To address this issue, we synthesized a novel biotinylated derivative of dimedone, keeping the dione cassette required for sulfenate reactivity but adding the functionality of a biotin tag. Biotin-amido(5-methyl-5-carboxamidocyclohexane 1,3-dione) tetragol (biotin dimedone) was prepared in six steps, combining 3,5-dimethoxybenzoic acid (Birch reduction, ultimately leading to the dimedone unit with a carboxylate functionality), 1-amino-11-azido-3,6,9-trioxaundecane (a differentially substituted tetragol spacer), and biotin. We loaded biotin dimedone (0.1 mM, 30 min) into rat ventricular myocytes, treated them with H 2 O 2 (0.1-10,000 M, 5 min), and monitored derivatization on Western blots using streptavidin-horseradish peroxidase. There was a dose-dependent increase in labeling of multiple proteins that was maximal at 0.1 or 1 mM H 2 O 2 and declined sharply below basal with 10 mM treatment. Cellwide labeling was observed in fixed cells probed with avidin-FITC using a confocal fluorescence microscope. Similar H 2 O 2 -induced labeling was observed in isolated rat hearts. Hearts loaded and subjected to hypoxia showed a striking loss of labeling, which returned when oxygen was resupplied, highlighting the protein sulfenates as oxygen sensors. Cardiac proteins that were sulfenated during oxidative stress were purified with avidin-agarose and identified by separation of tryptic digests by liquid chromatography with on-line analysis by mass spectrometry.